Lipids for use in lipid nanoparticle formulations

ABSTRACT

Compounds are provided having the following structure (I) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein X, Y, L1, L2, L3, G1, G2 and G3 are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND Technical Field

Embodiments of the present invention generally relate to novel lipidsthat can be used in combination with other lipid components, such asneutral lipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles for delivery of therapeutic agents, such as nucleic acids(e.g., oligonucleotides, messenger RNA), both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto affect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face the use of oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from lipids formulatedwith other lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved lipids and lipid nanoparticles for thedelivery of oligonucleotides. Preferably, these lipid nanoparticleswould provide optimal drug:lipid ratios, protect the nucleic acid fromdegradation and clearance in serum, be suitable for systemic or localdelivery, and provide intracellular delivery of the nucleic acid. Inaddition, these lipid-nucleic acid particles should be well-toleratedand provide an adequate therapeutic index, such that patient treatmentat an effective dose of the nucleic acid is not associated withunacceptable toxicity and/or risk to the patient. The present inventionprovides these and related advantages.

BRIEF SUMMARY

In brief, embodiments of the present invention provide lipid compounds,including stereoisomers, pharmaceutically acceptable salts, prodrugs ortautomers thereof, which can be used alone or in combination with otherlipid components such as neutral lipids, charged lipids, steroids(including for example, all sterols) and/or their analogs, and/orpolymer conjugated lipids to form lipid nanoparticles for the deliveryof therapeutic agents. In some instances, the lipid nanoparticles areused to deliver nucleic acids such as antisense and/or messenger RNA.Methods for use of such lipid nanoparticles for treatment of variousdiseases or conditions, such as those caused by infectious entitiesand/or insufficiency of a protein, are also provided.

In one embodiment, compounds having the following structure (I) areprovided:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein X, Y, L¹, L², L³, G¹, G² and G³ are as defined herein.

Pharmaceutical compositions comprising one or more of the foregoingcompounds of structure (I) and a therapeutic agent are also provided.Also provided are lipid nanoparticles (LNPs) comprising one or morecompounds of structure (I). In some embodiments, the pharmaceuticalcompositions and/or LNPs further comprise one or more componentsselected from neutral lipids, charged lipids, steroids and polymerconjugated lipids. The disclosed compositions are useful for formationof lipid nanoparticles for the delivery of the therapeutic agent.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing a composition of lipid nanoparticlescomprising the compound of structure (I) and a therapeutic agent anddelivering the composition to the patient. In some embodiments themethod for administering a therapeutic agent to a patient in needthereof comprises administering an LNP comprising one or more compoundsof structure (I) and the therapeutic agent to the patient.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand thatembodiments of the invention may be practiced without these details.

Embodiments of the present invention are based, in part, upon thediscovery of novel lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments the present invention provides nucleic acid-lipidnanoparticle compositions comprising one or more of the novel lipidsdescribed herein that provide increased activity of the nucleic acid andimproved tolerability of the compositions in vivo, resulting in asignificant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described. For example,embodiments provide a lipid nanoparticle comprising one or morecompounds of structure (I).

In particular embodiments, the present invention provides novel lipidsthat enable the formulation of improved compositions for the in vitroand in vivo delivery of mRNA and/or other oligonucleotides. In someembodiments, these improved lipid nanoparticle compositions are usefulfor expression of protein encoded by mRNA. In other embodiments, theseimproved lipid nanoparticles compositions are useful for upregulation ofendogenous protein expression by delivering miRNA inhibitors targetingone specific miRNA or a group of miRNA regulating one target mRNA orseveral mRNA. In other embodiments, these improved lipid nanoparticlecompositions are useful for down-regulating (e.g., silencing) theprotein levels and/or mRNA levels of target genes. In some otherembodiments, the lipid nanoparticles are also useful for delivery ofmRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antigen or antibody.

The lipid nanoparticles and compositions of embodiments of the presentinvention may be used for a variety of purposes, including the deliveryof encapsulated or associated (e.g., complexed) therapeutic agents suchas nucleic acids to cells, both in vitro and in vivo. Accordingly,embodiments of the present invention provide methods of treating orpreventing diseases or disorders in a subject in need thereof bycontacting the subject with a lipid nanoparticle that encapsulates or isassociated with a suitable therapeutic agent, wherein the lipidnanoparticle comprises one or more of the novel lipids described herein.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of embodiments of the presentinvention may be used to induce expression of a desired protein both invitro and in vivo by contacting cells with a lipid nanoparticlecomprising one or more novel lipids described herein, wherein the lipidnanoparticle encapsulates or is associated with a nucleic acid that isexpressed to produce the desired protein (e.g., a messenger RNA orplasmid encoding the desired protein) or inhibit processes thatterminate expression of mRNA (e.g., miRNA inhibitors). Alternatively,the lipid nanoparticles and compositions of embodiments of the presentinvention may be used to decrease the expression of target genes andproteins both in vitro and in vivo by contacting cells with a lipidnanoparticle comprising one or more novel lipids (e.g., a compound ofstructure (I)) described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an antisense oligonucleotide or small interferingRNA (siRNA)). The lipid nanoparticles and compositions of embodiments ofthe present invention may also be used for co-delivery of differentnucleic acids (e.g., mRNA and plasmid DNA) separately or in combination,such as may be useful to provide an effect requiring colocalization ofdifferent nucleic acids (e.g., mRNA encoding for a suitable genemodifying enzyme and DNA segment(s) for incorporation into the hostgenome).

Nucleic acids for use with embodiments of this invention may be preparedaccording to any available technique. For mRNA, the primary methodologyof preparation is, but not limited to, enzymatic synthesis (also termedin vitro transcription) which currently represents the most efficientmethod to produce long sequence-specific mRNA. In vitro transcriptiondescribes a process of template-directed synthesis of RNA molecules froman engineered DNA template comprised of an upstream bacteriophagepromoter sequence (e.g., including but not limited to that from the T7,T3 and SP6 coliphage) linked to a downstream sequence encoding the geneof interest. Template DNA can be prepared for in vitro transcriptionfrom a number of sources with appropriate techniques which are wellknown in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012)

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies) as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g., Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v. 41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNAoligos, etc.). Techniques for the isolation of the mRNA transcripts arewell known in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukavsky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see, e.g., Kariko, K., Muramatsu, H.,Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA fortherapy: HPLC purification eliminates immune activation and improvestranslation of nucleoside-modified, protein-encoding mRNA, Nucl AcidRes, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K.,HPLC Purification of in vitro transcribed long RNA in SyntheticMessenger RNA and Cell Metabolism Modulation in Methods in MolecularBiology v. 969 (Rabinovich, P. H. Ed), 2013). HPLC purified mRNA hasbeen reported to be translated at much greater levels, particularly inprimary cells and in vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e. cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half-life, reduced susceptibility to 5′endonucleases, and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see, e.g., Grudzien-Nogalska, E.,Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E.,Sahin, U., Jemielity, J., and Rhoads, R. E., Synthetic mRNAs withsuperior translation and stability properties in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogeneous length. 5′-capping and 3′-poly (A)tailing can be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(Epicenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see e.g. Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v. 10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko,K., In vitro transcription of long RNA containing modified nucleosidesin Synthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v. 969 (Rabinovich, P. H. Ed), 2013); Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v. 16, 1833-1840. The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see e.g. US2012/0251618). In vitro synthesis of nucleoside-modifiedmRNA have been reported to have reduced ability to activate immunesensors with a concomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see e.g. Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used forembodiments of this invention. For oligonucleotides, methods ofpreparation include but are not limited to chemical synthesis andenzymatic, chemical cleavage of a longer precursor, in vitrotranscription as described above, etc. Methods of synthesizing DNA andRNA nucleotides are widely used and well known in the art (see, e.g.Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn,P. (ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

For plasmid DNA, preparation for use with embodiments of this inventioncommonly utilizes but is not limited to expansion and isolation of theplasmid DNA in vitro in a liquid culture of bacteria containing theplasmid of interest. The presence of a gene in the plasmid of interestthat encodes resistance to a particular antibiotic (penicillin,kanamycin, etc.) allows those bacteria containing the plasmid ofinterest to selectively grow in antibiotic-containing cultures. Methodsof isolating plasmid DNA are widely used and well known in the art (see,e.g. Heilig, J., Elbing, K. L. and Brent, R (2001) Large-ScalePreparation of Plasmid DNA. Current Protocols in Molecular Biology.41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillström, S.,Björnestedt, R. and Schmidt, S. R. (2008), Large-scale production ofendotoxin-free plasmids for transient expression in mammalian cellculture. Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No.6,197,553B1). Plasmid isolation can be performed using a variety ofcommercially available kits including, but not limited to Plasmid Plus(Qiagen), GenJET plasmid MaxiPrep (Thermo) and PureYield MaxiPrep(Promega) kits as well as with commercially available reagents.

Various exemplary embodiments of the lipids of the present invention,lipid nanoparticles and compositions comprising the same, and their useto deliver active (e.g. therapeutic agents), such as nucleic acids, tomodulate gene and protein expression, are described in further detailbelow.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g. asample of cells in culture expressing the desired protein) or a testmammal (e.g. a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid (e.g. nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g. a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal model such as a rodent (e.g. mouse) or non-humanprimate (e.g. monkey) model) that is not contacted with or administeredthe nucleic acid. When the desired protein is present in a controlsample or a control mammal, the expression of a desired protein in acontrol sample or a control mammal may be assigned a value of 1.0. Inparticular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g. a sample of cells in culture expressing the target gene) or a testmammal (e.g. a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g. monkey) model) iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g. a sample of cells in culture expressing the targetgene) or a control mammal (e.g. a mammal such as a human or an animalmodel such as a rodent (e.g. mouse) or non-human primate (e.g. monkey)model) that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g. an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge. Preferred cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range e.g. pH 3 topH 9. Charged lipids may be synthetic or naturally derived. Examples ofcharged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates,dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g. DC-Chol).

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of structure (I) or other specifiedcationic lipids. In some embodiments, lipid nanoparticles are includedin a formulation that can be used to deliver an active agent ortherapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, tumor, and the like). In someembodiments, the lipid nanoparticles of the invention comprise a nucleicacid. Such lipid nanoparticles typically comprise a compound ofstructure (I) and one or more excipient selected from neutral lipids,charged lipids, steroids and polymer conjugated lipids. In someembodiments, the active agent or therapeutic agent, such as a nucleicacid, may be encapsulated in the lipid portion of the lipid nanoparticleor an aqueous space enveloped by some or all of the lipid portion of thelipid nanoparticle, thereby protecting it from enzymatic degradation orother undesirable effects induced by the mechanisms of the host organismor cells e.g. an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are disclosed in, e.g.,U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub.Nos. WO 2017/004143, WO 2015/199952, WO 2013/016058 and WO 2013/086373,the full disclosures of which are herein incorporated by reference intheir entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated,having, for example, from one to twenty-four carbon atoms (C₁-C₂₄alkyl), six to twenty-four carbon atoms (C₆-C₂₄ alkyl), four to twentycarbon atoms (C₄-C₂₀ alkyl), six to sixteen carbon atoms (C₆-C₁₆ alkyl),six to nine carbon atoms (C₆-C₉ alkyl), one to fifteen carbon atoms(C₁-C₁₅ alkyl), one to twelve carbon atoms (C₁-C₁₂ alkyl), one to eightcarbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆ alkyl) andwhich is attached to the rest of the molecule by a single bond, e.g.,methyl, ethyl, n-propyl, 1 methylethyl (iso propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t butyl), 3-methylhexyl, 2-methylhexyl, and the like.Unless stated otherwise specifically in the specification, an alkylgroup is optionally substituted.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which contains one ormore carbon-carbon double bonds, and having, for example, from two totwenty-four carbon atoms (C₂-C₂₄ alkenyl), six to twenty-four carbonatoms (C₆-C₂₄ alkenyl), four to twenty carbon atoms (C₄-C₂₀ alkenyl),six to sixteen carbon atoms (C₆-C₁₆ alkenyl), six to nine carbon atoms(C₆-C₉ alkenyl), two to fifteen carbon atoms (C₂-C₁₅ alkenyl), two totwelve carbon atoms (C₂-C₁₂ alkenyl), two to eight carbon atoms (C₂-C₈alkenyl) or two to six carbon atoms (C₂-C₆ alkenyl) and which isattached to the rest of the molecule by a single bond, e.g., ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like.Unless stated otherwise specifically in the specification, an alkenylgroup is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated, andhaving, for example, from one to twenty-four carbon atoms (C₁-C₂₄alkylene), one to fifteen carbon atoms (C₁-C₁₅ alkylene), one to twelvecarbon atoms (C₁-C₁₂ alkylene), one to eight carbon atoms (C₁-C₈alkylene), one to six carbon atoms (C₁-C₆ alkylene), two to four carbonatoms (C₂-C₄ alkylene), one to two carbon atoms (C₁-C₂ alkylene), e.g.,methylene, ethylene, propylene, n-butylene, and the like. The alkylenechain is attached to the rest of the molecule through a single bond andto the radical group through a single bond. The points of attachment ofthe alkylene chain to the rest of the molecule and to the radical groupcan be through one carbon or any two carbons within the chain. Unlessstated otherwise specifically in the specification, an alkylene chainmay be optionally substituted.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, which contains one ormore carbon-carbon double bonds, and having, for example, from two totwenty-four carbon atoms (C₂-C₂₄ alkenylene), two to fifteen carbonatoms (C₂-C₁₅ alkenylene), two to twelve carbon atoms (C₂-C₁₂alkenylene), two to eight carbon atoms (C₂-C₈ alkenylene), two to sixcarbon atoms (C₂-C₆ alkenylene) or two to four carbon atoms (C₂-C₄alkenylene), e.g., ethenylene, propenylene, n-butenylene, and the like.The alkenylene chain is attached to the rest of the molecule through asingle or double bond and to the radical group through a single ordouble bond. The points of attachment of the alkenylene chain to therest of the molecule and to the radical group can be through one carbonor any two carbons within the chain. Unless stated otherwisespecifically in the specification, an alkenylene chain may be optionallysubstituted.

“Heteroalkylene” refers to an alkylene as defined herein, wherein atleast one carbon-carbon bond within the alkylene is replaced by acarbon-heteoratom-carbon bond. Heteroatoms include O, N and S. Anexemplary heteroalkylene is aminylheteroalkylene, which is aheteroalkylene wherein the heteroatom is N (e.g., —NR—, wherein R is Hor C₁-C₁₂ alkyl). Other heteroalkylenes include alkylene oxides, such asethylene oxides and polyethylene oxides. Heteroalkylenes include one totwenty-four carbon atoms (C₁-C₂₄ heteroalkylene), one to fifteen carbonatoms (C₁-C₁₅ heteroalkylene), one to twelve carbon atoms (C₁-C₁₂heteroalkylene), one to eight carbon atoms (C₁-C₈ heteroalkylene), oneto six carbon atoms (C₁-C₆ heteroalkylene), two to four carbon atoms(heteroalkylene), one to two carbon atoms (C₁-C₂ heteroalkylene), andthe like. The heteroalkylene chain is attached to the rest of themolecule through a single bond and to the radical group through a singlebond. The points of attachment of the heteroalkylene chain to the restof the molecule and to the radical group can be through one carbon orany two carbons within the chain. Unless stated otherwise specificallyin the specification, a heteroalkylene chain may be optionallysubstituted.

“Heteroalkenylene” is a heteroalkylene as defined herein, comprising atleast on carbon-carbon double bond. Heteroalkenylenes include from twoto twenty-four carbon atoms (C₂-C₂₄ heteroalkenylene), two to fifteencarbon atoms (C₂-C₁₅ heteroalkenylene), two to twelve carbon atoms(C₂-C₁₂ heteroalkenylene), two to eight carbon atoms (C₂-C₈heteroalkenylene), two to six carbon atoms (C₂-C₆ heteroalkenylene) ortwo to four carbon atoms (C₂-C₄ heteroalkenylene), and the like. Theheteroalkenylene chain is attached to the rest of the molecule through asingle or double bond and to the radical group through a single ordouble bond. The points of attachment of the heteroalkenylene chain tothe rest of the molecule and to the radical group can be through onecarbon or any two carbons within the chain. Unless stated otherwisespecifically in the specification, a heteroalkenylene chain may beoptionally substituted.

The term “substituted” used herein means any of the above groups (e.g.alkyl, alkenyl, alkylene, alkenylene, heteroalkylene andheteroalkenylene) wherein at least one hydrogen atom is replaced by abond to a non-hydrogen atom such as, but not limited to: a halogen atomsuch as F, Cl, Br, or I; oxo groups (═O); hydroxyl groups (—OH); C₁-C₁₂alkyl groups; cycloalkyl groups; —(C═O)OR′; —O(C═O)R′; —C(═O)R′; —OR′;—S(O)_(x)R′; —S—SR′; —C(═O)SR′; —SC(═O)R′; —NR′R′; —NR′C(═O)R′;—C(═O)NR′R′; —NR′C(═O)NR′R′; —OC(═O)NR′R′; —NR′C(═O)OR′;—NRS(O)_(x)NR′R′; —NR′S(O)_(x)R′; and —S(O)_(x)NR′R′, wherein: R′ is, ateach occurrence, independently H, C₁-C₁₅ alkyl or cycloalkyl, and x is0, 1 or 2. In some embodiments the substituent is a C₁-C₁₂ alkyl group.In other embodiments, the substituent is a cycloalkyl group. In otherembodiments, the substituent is a halo group, such as fluoro. In otherembodiments, the substituent is an oxo group. In other embodiments, thesubstituent is a hydroxyl group. In other embodiments, the substituentis an alkoxy group (—OR′). In other embodiments, the substituent is acarboxyl group. In other embodiments, the substituent is an amine group(—NR′R′).

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of structure (I). Thus, the term “prodrug” refers to ametabolic precursor of a compound of structure (I) that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of structure (I). Prodrugs are typically rapidly transformed invivo to yield the parent compound of structure (I), for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of structure (I) in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of structure (I) may be prepared by modifying functional groupspresent in the compound of structure (I) in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of structure (I). Prodrugs include compounds ofstructure (I) wherein a hydroxy, amino or mercapto group is bonded toany group that, when the prodrug of the compound of structure (I) isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of structure (I) and the like.

Embodiments of the invention disclosed herein are also meant toencompass all pharmaceutically acceptable compounds of the compound ofstructure (I) being isotopically-labelled by having one or more atomsreplaced by an atom having a different atomic mass or mass number.Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C,¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I,respectively. These radiolabeled compounds could be useful to helpdetermine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to pharmacologically important site of action. Certainisotopically-labelled compounds of structure (I) or (II), for example,those incorporating a radioactive isotope, are useful in drug and/orsubstrate tissue distribution studies. The radioactive isotopes tritium,i.e., ³H, and carbon-14, i.e., ¹⁴C, are particularly useful for thispurpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed in the Preparations and Examples as set out below using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously employed.

Embodiments of the invention disclosed herein are also meant toencompass the in vivo metabolic products of the disclosed compounds.Such products may result from, for example, the oxidation, reduction,hydrolysis, amidation, esterification, and the like of the administeredcompound, primarily due to enzymatic processes. Accordingly, embodimentsof the invention include compounds produced by a process comprisingadministering a compound of this invention to a mammal for a period oftime sufficient to yield a metabolic product thereof. Such products aretypically identified by administering a radiolabeled compound ofstructure (I) in a detectable dose to an animal, such as rat, mouse,guinea pig, monkey, or to human, allowing sufficient time for metabolismto occur, and isolating its conversion products from the urine, blood orother biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of structure(I). As used herein, the term “solvate” refers to an aggregate thatcomprises one or more molecules of a compound of structure (I) with oneor more molecules of solvent. The solvent may be water, in which casethe solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. In some embodiments, the compound ofstructure (I) may exist as a true solvate, while in other cases, thecompound of structure (I) may merely retain adventitious water or be amixture of water plus some adventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofstructure (I) and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of structure (I) which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle ofembodiments the invention which constitutes a “therapeutically effectiveamount” will vary depending on the compound, the condition and itsseverity, the manner of administration, and the age of the mammal to betreated, but can be determined routinely by one of ordinary skill in theart having regard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment; (iii) relieving the disease or condition, i.e., causingregression of the disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of structure (I), or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. Embodiments of the present invention aremeant to include all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

Compounds

In an aspect, the invention provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with oligonucleotides. Without wishing to be bound bytheory, it is thought that these lipid nanoparticles shieldoligonucleotides from degradation in the serum and provide for effectivedelivery of oligonucleotides to cells in vitro and in vivo.

In one embodiment, the compounds have the following structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein:

X is N, and Y is absent; or X is CR, and Y is NR;

L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,—C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c),—NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;

L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR²,—C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),—NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or a directbond to R²;

L³ is —O(C═O)R³ or —(C═O)OR³;

G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene;

G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₁-C₂₄ heteroalkylene orC₂-C₂₄ heteroalkenylene;

R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂ alkylor C₁-C₁₂ alkenyl;

R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl;

each R is independently H or Q-C₁₂ alkyl;

R¹, R² and R³ are each independently C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl; and

x is 0, 1 or 2, and

wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene andheteroalkenylene is independently substituted or unsubstituted unlessotherwise specified.

In more embodiments of structure (I):

X is N, and Y is absent; or X is CR, and Y is NR;

L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,—C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c),—NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹;

L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR²,—C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),—NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or a directbond to R²;

L³ is —O(C═O)R³ or —(C═O)OR³;

G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene;

G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₁-C₂₄ heteroalkylene orC₂-C₂₄ heteroalkenylene when X is CR, and Y is NR; and G³ is C₁-C₂₄heteroalkylene or C₂-C₂₄ heteroalkenylene when X is N, and Y is absent;

R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂ alkylor C₁-C₁₂ alkenyl;

R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl;

each R is independently H or C₁-C₁₂ alkyl;

R¹, R² and R³ are each independently C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl; and

x is 0, 1 or 2, and

wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene andheteroalkenylene is independently substituted or unsubstituted unlessotherwise specified.

In other embodiments of structure (I):

X is N and Y is absent, or X is CR and Y is NR;

L¹ is —O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹,—C(═O)SR¹, —SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c),—NR^(a)C(═O)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR′;

L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR²,—C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f),—NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or a directbond to R²;

L³ is —O(C═O)R³ or —(C═O)OR³;

G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene;

G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₁-C₂₄ heteroalkylene orC₂-C₂₄ heteroalkenylene;

R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂ alkylor C₁-C₁₂ alkenyl;

R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl;

each R is independently H or C₁-C₁₂ alkyl;

R¹, R² and R³ are each independently branched C₆-C₂₄ alkyl or branchedC₆-C₂₄ alkenyl; and

x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene,heteroalkylene and heteroalkenylene is independently substituted orunsubstituted unless otherwise specified.

In certain embodiments, G³ is unsubstituted. In more specificembodiments G³ is C₂-C₁₂ alkylene, tor example, in some embodiments G³is C₃-C₇ alkylene or in other embodiments G is C₃-C₁₂ alkylene. In someembodiments, G is C₂ or C₃ alkylene.

In other embodiments, G³ is C₁-C₁₂ heteroalkylene, for example C₁-C₁₂aminylalkylene.

In certain embodiments, X is N and Y is absent. In other embodiments, Xis CR and Y is NR, for example in some of these embodiments R is H.

In some of the foregoing embodiments, the compound has one of thefollowing structures (IA), (IB), (IC) or (ID):

In some of the foregoing embodiments, L¹ is —O(C═O)R¹, —(C═O)OR¹ or—C(═O)NR^(b)R^(c), and L² is —O(C═O)R², —(C═O)OR² or —C(═O)NR^(e)R^(f).In other specific embodiments, L¹ is —(C═O)OR′ and L² is —(C═O)OR². Inany of the foregoing embodiments, L³ is —(C═O)OR³.

In some of the foregoing embodiments, G¹ and G² are each independentlyC₂-C₁₂ alkylene, for example C₄-C₁₀ alkylene.

In some of the foregoing embodiments, R¹, R² and R³ are each,independently branched C₆-C₂₄ alkyl. For example, in some embodiments,R¹, R² and R³ each, independently have the following structure:

wherein:

R^(7a) and R^(7b) are, at each occurrence, independently H or C₁-C₁₂alkyl; and

a is an integer from 2 to 12, wherein R^(7a), R^(7b) and a are eachselected such that R¹ and R² each independently comprise from 6 to 20carbon atoms. For example, in some embodiments a is an integer rangingfrom 5 to 9 or from 8 to 12.

In some of the foregoing embodiments, at least one occurrence of R^(7a)is H. For example, in some embodiments, R^(7a) is H at each occurrence.In other different embodiments of the foregoing, at least one occurrenceof R^(7b) is C₁-C₈ alkyl. For example, in some embodiments, C₁-C₈ alkylis methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl,n-hexyl or n-octyl.

In some of the foregoing embodiments, X is CR, Y is NR and R³ is C₁-C₁₂alkyl, such as ethyl, propyl or butyl. In some of these embodiments, R¹and R² are each independently branched C₆-C₂₄ alkyl.

In different embodiments, R¹, R² and R³ each, independently have one ofthe following structures:

In certain embodiments, R¹ and R² and R³ are each, independently,branched C₆-C₂₄ alkyl and R³ is C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl.

In some of the foregoing embodiments, R^(b), R^(c), R^(e) and R^(f) areeach independently C₃-C₁₂ alkyl. For example, in some embodiments R^(b),R^(c), R^(e) and R^(f) are n-hexyl and in other embodiments R^(b),R^(c), R^(e) and R^(f) are n-octyl.

In various different embodiments, the compound has one of the structuresset forth in Table 1 below.

TABLE 1 Representative Compounds No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

The compounds in Table 1 were prepared and tested according to methodsknown in the art, for example those general methods described hereinbelow.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific substituent and/or variable in thecompound structure (I), as set forth above, may be independentlycombined with other embodiments and/or substituents and/or variables ofcompounds of structure (I) to form embodiments of the inventions notspecifically set forth above. In addition, in the event that a list ofsubstituents and/or variables is listed for any particular R group, Lgroup or G group in a particular embodiment and/or claim, it isunderstood that each individual substituent and/or variable may bedeleted from the particular embodiment and/or claim and that theremaining list of substituents and/or variables will be considered to bewithin the scope of embodiments of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In some embodiments, compositions comprising any one or more of thecompounds of structure (I) and a therapeutic agent are provided. In someembodiments are provided a lipid nanoparticle comprising one or morecompounds of structure (I). For example, in some embodiments, thecompositions comprise any of the compounds of structure (I) and atherapeutic agent and one or more excipient selected from neutrallipids, steroids and polymer conjugated lipids. Other pharmaceuticallyacceptable excipients and/or carriers are also included in variousembodiments of the compositions.

In some embodiments, the neutral lipid is selected from DSPC, DPPC,DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid isDSPC. In various embodiments, the molar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 5:1 to 1:1.

In various embodiments, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide(PEG-cer), or a PEG dialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 20:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R⁸ and R⁹ are each independently a straight or branched, alkyl, alkenylor alkynyl containing from 10 to 30 carbon atoms, wherein the alkyl,alkenyl or alkynyl is optionally interrupted by one or more ester bonds;and

w has a mean value ranging from 30 to 60.

In some embodiments, R⁸ and R⁹ are each independently straight alkylcontaining from 12 to 16 carbon atoms. In some embodiments, w has a meanvalue ranging from 43 to 53. In other embodiments, the average w isabout 45. In other different embodiments, the average w is about 49.

In some embodiments, lipid nanoparticles (LNPs) comprising any one ormore of the compounds of structure (I) and a therapeutic agent areprovided. For example, in some embodiments, the LNPs comprise any of thecompounds of structure (I) and a therapeutic agent and one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids.

In some embodiments of the LNPs, the neutral lipid is selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, theneutral lipid is DSPC. In various embodiments, the molar ratio of thecompound to the neutral lipid ranges from about 2:1 to about 8:1.

In various embodiments of the LNPs, the compositions further comprise asteroid or steroid analogue. In certain embodiments, the steroid orsteroid analogue is cholesterol. In some of these embodiments, the molarratio of the compound to cholesterol ranges from about 5:1 to 1:1.

In various embodiments of the LNPs, the polymer conjugated lipid is apegylated lipid. For example, some embodiments include a pegylateddiacylglycerol (PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 20:1.

In some embodiments, the LNPs comprise a pegylated lipid having thefollowing structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R⁸ and R⁹ are each independently a straight or branched, alkyl, alkenylor alkynyl containing from 10 to 30 carbon atoms, wherein the alkyl,alkenyl or alkynyl is optionally interrupted by one or more ester bonds;and

w has a mean value ranging from 30 to 60.

In some embodiments, R⁸ and R⁹ are each independently straight alkylcontaining from 12 to 16 carbon atoms. In some embodiments, w has a meanvalue ranging from 43 to 53. In other embodiments, the average w isabout 45. In other different embodiments, the average w is about 49.

Preparation methods for the above lipids, lipid nanoparticles andcompositions are described herein below and/or known in the art, forexample, in PCT Pub. No. WO 2015/199952, WO 2017/004143 and WO2017/075531, each of which is incorporated herein by reference in theirentireties.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense and messenger RNA.

In other different embodiments, the invention is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoingcompositions and administering the composition to the patient

For the purposes of administration, the compounds of structure (I)(typically in the form of lipid nanoparticles in combination with atherapeutic agent) may be administered as a raw chemical or may beformulated as pharmaceutical compositions. Pharmaceutical compositionsof embodiments of the present invention comprise a compound of structure(I) (e.g., as a component in an LNP) and one or more pharmaceuticallyacceptable carrier, diluent or excipient. The compound of structure (I)is present in the composition in an amount which is effective to form alipid nanoparticle and deliver the therapeutic agent, e.g., for treatinga particular disease or condition of interest. Appropriateconcentrations and dosages can be readily determined by one skilled inthe art.

Administration of the compositions and/or LNPs of embodiments of theinvention can be carried out via any of the accepted modes ofadministration of agents for serving similar utilities. Thepharmaceutical compositions of embodiments of the invention may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suspensions, suppositories, injections, inhalants, gels,microspheres, and aerosols. Typical routes of administering suchpharmaceutical compositions include, without limitation, oral, topical,transdermal, inhalation, peritoneal, sublingual, buccal, rectal,vaginal, and intranasal. The term peritoneal as used herein includessubcutaneous injections, intravenous, intramuscular, intradermal,intrasternal injection or infusion techniques. Pharmaceuticalcompositions of the invention are formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa subject or patient take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of acompound of structure (I) in aerosol form may hold a plurality of dosageunits. Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in this art; for example, see Remington;The Science and Practice of Pharmacy, 20th Edition (Philadelphia Collegeof Pharmacy and Science, 2000). The composition to be administered will,in any event, contain a therapeutically effective amount of a compoundof structure (I), or a pharmaceutically acceptable salt thereof, fortreatment of a disease or condition of interest in accordance with theteachings of embodiments of this invention.

A pharmaceutical composition of embodiments of the invention may be inthe form of a solid or liquid. In one aspect, the carrier(s) areparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) may be liquid, with the compositions being,for example, oral syrup, injectable liquid or an aerosol, which isuseful in, for example, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds or LNPs, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of embodiments of the invention,whether they be solutions, suspensions or other like form, may includeone or more of the following adjuvants: sterile diluents such as waterfor injection, saline solution, preferably physiological saline,Ringer's solution, isotonic sodium chloride, fixed oils such assynthetic mono or diglycerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose; agents to act ascryoprotectants such as sucrose or trehalose. The peritoneal preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Physiological saline is a preferred adjuvant.An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of embodiments of the inventionintended for either peritoneal or oral administration should contain anamount of a compound of structure (I) such that a suitable LNP will beobtained.

The pharmaceutical composition of embodiments of the invention may beintended for topical administration, in which case the carrier maysuitably comprise a solution, emulsion, ointment or gel base. The base,for example, may comprise one or more of the following: petrolatum,lanolin, polyethylene glycols, bee wax, mineral oil, diluents such aswater and alcohol, and emulsifiers and stabilizers. Thickening agentsmay be present in a pharmaceutical composition for topicaladministration. If intended for transdermal administration, thecomposition may include a transdermal patch or iontophoresis device.

The pharmaceutical composition of embodiments of the invention may beintended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Thecomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include, withoutlimitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of embodiments of the invention mayinclude various materials, which modify the physical form of a solid orliquid dosage unit. For example, the composition may include materialsthat form a coating shell around the active ingredients. The materialsthat form the coating shell are typically inert, and may be selectedfrom, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of embodiments of the invention in solidor liquid form may include an agent that binds to the compound ofstructure (I) and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include a monoclonal orpolyclonal antibody, or a protein.

The pharmaceutical composition of embodiments of the invention mayconsist of dosage units that can be administered as an aerosol. The termaerosol is used to denote a variety of systems ranging from those ofcolloidal nature to systems consisting of pressurized packages. Deliverymay be by a liquefied or compressed gas or by a suitable pump systemthat dispenses the active ingredients. Aerosols of compounds ofstructure (I) may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,sub-containers, and the like, which together may form a kit. One skilledin the art, without undue experimentation may determine preferredaerosols.

The pharmaceutical compositions of embodiments of the invention may beprepared by methodology well known in the pharmaceutical art. Forexample, a pharmaceutical composition intended to be administered byinjection can be prepared by combining the lipid nanoparticles of theinvention with sterile, distilled water or other carrier so as to form asolution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the compound of structure (I) so as tofacilitate dissolution or homogeneous suspension of the compound in theaqueous delivery system.

The compositions of embodiments of the invention, or theirpharmaceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the activity of the specific therapeutic agent employed; themetabolic stability and length of action of the therapeutic agent; theage, body weight, general health, sex, and diet of the patient; the modeand time of administration; the rate of excretion; the drug combination;the severity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of embodiments of the invention may also be administeredsimultaneously with, prior to, or after administration of one or moreother therapeutic agents. Such combination therapy includesadministration of a single pharmaceutical dosage formulation of acomposition of embodiments of the invention and one or more additionalactive agents, as well as administration of the composition of theinvention and each active agent in its own separate pharmaceuticaldosage formulation. For example, a composition of embodiments of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof structure (I) and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of structure(I) which are pharmacologically active. Such derivatives may thereforebe described as “prodrugs”. All prodrugs of compounds of structure (I)are included within the scope of embodiments of the invention.

Furthermore, all compounds of structure (I) which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds ofstructure (I) can be converted to their free base or acid form bystandard techniques.

The compounds of structure (I), and lipid nanoparticles comprising thesame, can be prepared according to methods known or derivable by one ofordinary skill in the art, for example methods analogous to thosedisclosed in PCT Pub. No. WO 2015/199952, WO 2017/004143 and WO2017/075531, each of which is incorporated herein by reference in theirentireties.

The following General Reaction Schemes illustrate exemplary methods tomake compounds of structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein X, Y, L¹, L², L³, G¹, G² and G³ are as defined herein. It isunderstood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of structure (I) not specifically illustrated below by usingthe appropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

Embodiments of the compound of structure (I) (e.g., compound A-5) can beprepared according to General Reaction Scheme 1 (“Method A”), whereineach R independently represents R¹ or R², and each n is independently aninteger from 2 to 12. Referring to General Reaction Scheme 1, compoundsof structure A-1 can be purchased from commercial sources or preparedaccording to methods familiar to one of ordinary skill in the art. Amixture of A-1, A-2 and DMAP is treated with DCC to give the bromideA-3. A mixture of the bromide A-3, a base (e.g.,N,N-diisopropylethylamine) and A-4 is heated at a temperature and timesufficient to produce A-5 after any necessary workup and or purificationstep. Protecting group strategies or alternative order of syntheticsteps may be employed to avoid unwanted side reactions with the L³moiety as needed.

Embodiments of the compound of structure (I) (e.g., compound B-5) can beprepared according to General Reaction Scheme 2 (“Method B”), whereineach R independently represents R¹ or R², and each n is independently aninteger from 2 to 12. As shown in General Reaction Scheme 2, compoundsof structure B-1 can be purchased from commercial sources or preparedaccording to methods familiar to one of ordinary skill in the art. Asolution of B-1 (1 equivalent) is treated with acid chloride B-2 (1equivalent) and a base (e.g., triethylamine). The crude product istreated with an oxidizing agent (e.g., pyridinum chlorochromate) andintermediate product B-3 is recovered. A solution of crude B-3, an acid(e.g., acetic acid), and A-4 is then treated with a reducing agent(e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessarywork up and/or purification. Protecting group strategies or alternativeorder of synthetic steps may be employed to avoid unwanted sidereactions with the L moiety as needed.

Embodiments of the compound of structure (I) can be prepared accordingto General Reaction Scheme 3 (“Method C”), wherein each G independentlyrepresents G¹, G^(1′), G² or G^(2′), and each L independently representsL¹, L^(1′), L² or L^(2′). Referring to General Reaction Scheme 1,compounds of structure A-4 and C-1 can be purchased from commercialsources or prepared according to methods familiar to one of ordinaryskill in the art. A solution of A-4 and C-1 is treated with a reducingagent (e.g., sodium triacetoxyborohydride) to obtain a compound ofstructure (I) after any necessary work up.

It should be noted that although starting materials A-1 and B-1 aredepicted above as including only saturated methylene carbons, startingmaterials which include carbon-carbon double bonds may also be employedfor preparation of compounds which include carbon-carbon double bonds.

It should be noted that various alternative strategies for preparationof compounds of structure (I) are available to those of ordinary skillin the art. For example, certain moieties may include a substituent,such as hydroxyl, and appropriate protecting groups may be required tomask the substituent, or the substituent may be added at a differentpoint in the synthesis to avoid unwanted side reactions. The use ofprotecting groups as needed and other modification to the above GeneralReaction Schemes 1-3 will be readily apparent to one of ordinary skillin the art. The following examples are provided for purpose ofillustration and not limitation.

Example 1 Luciferase mRNA In Vivo Evaluation Using Lipid NanoparticleCompositions

A lipid of structure (I), DSPC, cholesterol and PEG-lipid weresolubilized in ethanol at a molar ratio of 50:10:38.5:1.5 or47.5:10:40.8:1.7. Lipid nanoparticles (LNP) were prepared at a totallipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, themRNA was diluted to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4.Syringe pumps were used to mix the ethanolic lipid solution with themRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) withtotal flow rates above 15 mL/min. The ethanol was then removed and theexternal buffer replaced with PBS by dialysis. Finally, the lipidnanoparticles were filtered through a 0.2 μm pore sterile filter.

Studies were performed in 6-8 week old female C₅₇BL/6 mice (CharlesRiver) 8-10 week old CD-1 (Harlan) mice (Charles River) according toguidelines established by an institutional animal care committee (ACC)and the Canadian Council on Animal Care (CCAC). Varying doses ofmRNA-lipid nanoparticle were systemically administered by tail veininjection and animals euthanized at a specific time point (e.g., 4hours) post-administration. Liver and spleen were collected inpre-weighed tubes, weights determined, immediately snap frozen in liquidnitrogen and stored at −80° C. until processing for analysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) was added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature was added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford, Ill.).Relative luminescence units (RLU) were then normalized to total μgprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega).

The FLuc mRNA (L-6107 or L-7602) from Trilink Biotechnologies willexpress a luciferase protein, originally isolated from the firefly,Photinus pyralis. FLuc is commonly used in mammalian cell culture tomeasure both gene expression and cell viability. It emitsbioluminescence in the presence of the substrate, luciferin. This cappedand polyadenylated mRNA is fully substituted with 5-methylcytidine andpseudouridine.

Example 2 Determination of Pk_(A) of Formulated Lipids

As described elsewhere, the pKa of formulated lipids is correlated withthe effectiveness of LNPs for delivery of nucleic acids (see Jayaramanet al, Angewandte Chemie, International Edition (2012), 51(34),8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). Insome embodiments, the preferred range of pKa is ˜5 to ˜7. The pK_(a) ofrepresentative lipids was determined in lipid nanoparticles using anassay based on fluorescence of 2-(p-toluidino)-6-napthalene sulfonicacid (TNS). Lipid nanoparticles comprising compound of structure(I)/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 or 47.5:10:40.8:1.7 mol%) in PBS at a concentration of 0.4 mM total lipid were prepared usingthe in-line process as described in Example 1. TNS was prepared as a 100μM stock solution in distilled water. Vesicles were diluted to 24 μMlipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES,10 mM ammonium acetate, and 130 mM NaCl, where the pH ranged from 2.5 to11. An aliquot of the TNS solution was added to give a finalconcentration of 1 μM and following vortex mixing fluorescence intensitywas measured at room temperature in a SLM Aminco Series 2 LuminescenceSpectrophotometer using excitation and emission wavelengths of 321 nmand 445 nm. A sigmoidal best fit analysis was applied to thefluorescence data and the pK_(a) was measured as the pH giving rise tohalf-maximal fluorescence intensity.

Lipid nanoparticle particle size was approximately 55-95 nm diameter,and in some instances approximately 70-90 nm diameter as determined byquasi-elastic light scattering using a Malvern Zetasizer Nano ZS(Malvern, UK). The diameters given are intensity weighted means.Encapsulation was determined using a fluorescent intercalating dye basedassay (Ribogreen).

Compounds of structure (I) were formulated using the following molarratio: 47.5% cationic lipid/10% distearoylphosphatidylcholine(DSPC)/40.8% Cholesterol/1.7% PEG lipid (“PEG-DMA” 2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide). Relativeactivity was determined by measuring luciferase expression in the liver4 hours following administration via tail vein injection as described inExample 1.

Example 3 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an In Vivo Luciferase mRNAExpression Rodent Model

The cationic lipids shown in Table 2 have previously been tested withnucleic acids. For comparative purposes, these lipids were also used toformulate lipid nanoparticles containing the FLuc mRNA (L-6107) using anin line mixing method, as described in Example 1 and in PCT/US10/22614,which is hereby incorporated by reference in its entirety. Lipidnanoparticles may be formulated using the following molar ratio: 50%Cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38.5%Cholesterol/1.5% PEG lipid (“PEG-DMG”, i.e.,1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with anaverage PEG molecular weight of 2000). In alternate embodiments,cationic lipid, DSPC, cholesterol and PEG-lipid are formulated at amolar ratio of approximately 47.5:10:40.8:1.7. Relative activity wasdetermined by measuring luciferase expression in the liver 4 hoursfollowing administration via tail vein injection as described inExample 1. The activity was compared at a dose of 0.3 and 1.0 mg mRNA/kgand expressed as ng luciferase/g liver measured 4 hours afteradministration, as described in Example 1.

TABLE 2 Comparator Lipids showing activity with mRNA Liver Luc Liver Luc@ 0.3 mg/kg @ 1.0 mg/kg Compound dose dose Structure MC2  4 ± 1 N/D

DLinDMA  13 ± 3  67 ± 20

MC4  41 ± 10 N/D

XTC2  80 ± 28 237 ± 99

MC3 198 ± 126 757 ± 528

319 (2% PEG) 258 ± 67 681 ± 203

137 281 ± 203 588 ± 303

A  77 ± 40 203 ± 122

Representative compounds of the invention shown in Table 3 wereformulated using the following molar ratio: 50% cationic lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG lipid(“PEG-DMA”2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide)or 47.5% cationic lipid/10% DSPC/40.8% Cholesterol/1.7% PEG lipid.Relative activity was determined by measuring luciferase expression inthe liver 4 hours following administration via tail vein injection asdescribed in Example 1. The activity was compared at a dose of 0.5 mgmRNA/kg unless noted otherwise and expressed as ng luciferase/g livermeasured 4 hours after administration, as described in Example 1.Compound numbers in Table 3 refer to the compound numbers of Table 1.

TABLE 3 Novel Cationic Lipids and Associated Activity Liver Luc @ 0.5mg/kg Cmp. (ng luc/ No. pK_(a) g liver) Structure 1 6.39 1235 ± 345*

2 5.76  29 ± 4

3 6.50  33 ± 1

4 6.11  102 ± 42

*dosed at 0.3 mg/kg

Example 4 Synthesis of Compound 1

Synthesis of 4-1

To a solution of 6-bromohexanoic acid (16 mmol, 3.12 g),2-hexyl-1-decanol (22.4 mmol, 5.43 g) and DMAP (8 mmol, 976 mg) in DCM(50 mL) was added DCC (17.6 mmol, 3.62 g). The resulting mixture wasstirred at RT for 16 h. The precipitate (DCU) was removed by filtration.The filtrate was concentrated and the resulting residue oil/solid waspurified by column chromatography on silica gel (0 to 5% ethyl acetatein hexanes). This gave the desired product as a colorless oil (5.79 g,colorless oil, 13.8 mmol, 86%).

Synthesis of 4-2

To a solution of 2-aminoethanol (333 mg, 5.46 mmol) in 35 ml ofanhydrous THF were added 4-1 (4.37 g, 10.4 mmol), potassium carbonate(1.44 g, 10.4 mmol), cesium carbonate (534 mg, 1.64 mmol) and sodiumiodide (30 mg). The resulting mixture in a sealed pressure flask washeated at 70 C for 6 days. The solvent was evaporated under reducedpressure and the residue was taken up in a mixture of hexane and ethylacetate (94:4) and washed with water and brine. The organic layer wasseparated and dried over anhydrous sodium sulfate. The dried extract(320 mL) was loaded on a column of silica gel. The column was eluted amixture of with Hexane, EtOAc and triethylamine (95:5:0 to 80:20:1).This gave the desired product as a colorless oil (2.68 g, 3.63 mmol,70%). ¹HNMR (400 MHz, CDCl₃) δ: 3.97 (d, 5.8 Hz, 4H), 3.53 (t, 5.3 Hz,2H), 3.08-2.79 (br. 1H), 2.57 (t, 5.3 Hz, 2H), 2.45 (t-like, 7.4 Hz,4H), 2.31 (t, 7.5 Hz, 4H), 1.67-1.59 (m, 6H), 1.51-1.41 (m, 4H),1.38-1.10 (52H), 0.89 (t-like, 6.8 Hz, 12H).

Synthesis of 4-3

To an ice-cooled solution of 027-76 (2.16 g, 2.93 mmol) in 8 mL ofCHCl₃, was added thionyl chloride (8.79 mmol, 1.05 g) in 35 mL ofchloroform dropwise under an Ar atmosphere. After the addition of SOCl₂(1-2 min) was complete, the ice bath was removed and the reactionmixture was stirred for 16 h at room temperature (20 C). Removal ofchloroform, and SOCl₂ under reduced pressure gave a thick dark red oil.The crude product was purified by flash column chromatography on silicagel (0 to 1% MeOH in chloroform with trace of Et₃N). The desired productwas obtained as brown oil (1.786 g, 2.36 mmol, 80%).

Synthesis of Compound 1

A solution of 4-3 (150 mg, 0.2 mmol), 4-4 (0.8 mmol, 240 mmol; preparedfrom 2-butyl-1-octanol, 6-Aminocaproic acid) andN,N-diisopropylethylamine (0.035 mL) in acetonitrile (10 mL) was sealedand was heated at 64 C overnight. The reaction mixture was cooled andconcentrated. The residue was purified twice by flash dry columnchromatography on silica gel (hexane-EtOAc-Et3N, 95:5:0 to 80:20:1 andMeOH in chloroform, 0 to 5%). The desired product was obtained ascolorless oil (36 mg). ¹HNMR (400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 6H),2.67-2.56 (m, 4H), 2.55-2.50 (m, 2H), 2.40 (t-like, 7.5 Hz, 4H),2.34-2.28 (m, 6H), 1.70-1.35 (m, 18H), 1.35-1.17 (m, 68H), 0.92-0.87 (m,18H).

Example 5 Synthesis of Compound 2

A solution of the ketone 5-1 (250 mg, 0.35 mmol; prepared according tothe literature procedure) and the amine 4-4 (0.35 mmol, 106 mg) in DCE(5 mL) was stirred at RT under Ar for about 15 min. To the solution wasadded sodium triacetoxyborohydride (1.06 mmol, 222 mg) and AcOH (0.49mmol, 29 mg). The mixture was stirred at RT under a Ar atmosphere for 7days. The reaction mixture was concentrated and diluted with a mixtureof hexanes and EtOAc (ca 19:1) and washed with dilute NaOH, sat NaHCO3and brine. The extract was dried over sodium sulfate and filteredthrough a pad of silica gel. The pad was washed with a mixture ofhexane-EtOAc-Et3N (80:20:1). The washing was concentrated to give thecrude product. The crude product was purified by flash dry columnchromatography on silica gel (MeOH in chloroform, 0 to 5%). The desiredproduct was obtained as colorless oil (111 mg, 0.11 mmol, 32%). ¹HNMR(400 MHz, CDCl3) δ: 3.97 (d, 5.8 Hz, 6H), 2.55 (t-like, 7.0 Hz, 2H),2.48-2.39 (m, 1H), 2.34-2.28 (m, 6H), 1.69-1.58 (m, 8H), 1.52-1.42 (m,3H), 1.42-1.17 (m, 78H), 0.92-0.87 (m, 18H), 0.80-0.72 (br., 1H).

Example 6 Synthesis of Compound 3

A solution of 6-2 (150 mg, 0.2 mmol), 6-1 (0.8 mmol, 284 mg; preparedfrom 2-hexyl-1-decanol, 6-Aminocaproic acid) andN,N-diisopropylethylamine (0.035 mL) in acetonitrile (10 mL) was sealedand was heated at 74 C overnight. The reaction mixture was cooled andconcentrated. The residue was purified twice by flash dry columnchromatography on silica gel (hexane-EtOAc-Et3N, 95:5:0 to 80:20:1 andMeOH in chloroform, 0 to 5%). The desired product was obtained ascolorless oil (96 mg, 0.09 mmol, 45%). 1HNMR (400 MHz, CDCl3) d: 3.97(d, 5.8 Hz, 6H), 2.67-2.56 (m, 4H), 2.55-2.50 (m, 2H), 2.40 (t-like, 7.5Hz, 4H), 2.34-2.28 (m, 6H), 1.70-1.35 (m, 18H), 1.35-1.17 (m, 76H),0.92-0.87 (m, 18H).

Example 7 Synthesis of Compound 4

A solution of the ketone 5-1 (250 mg, 0.35 mmol; prepared according tothe literature procedure) and the amine 7-1 (0.5 mmol, 122 mg; preparedfrom 2-ethyl-1-butanol, 6-Aminocaproic acid) in DCE (5 mL) was stirredat RT under Ar for about 15 min. To the solution was added sodiumtriacetoxyborohydride (1.06 mmol, 222 mg) and AcOH (0.49 mmol, 29 mg).The mixture was stirred at RT under a Ar atmosphere for 2 days. Thereaction mixture was concentrated and diluted with a mixture of hexanesand EtOAc (ca 19:1) and washed with dilute NaOH, sat NaHCO3 and brine.The extract was dried over sodium sulfate and filtered through a pad ofsilica gel. The pad was washed with a mixture of hexane-EtOAc-Et3N(80:20:1). The washing was concentrated to give the crude product. Thecrude product was purified by flash dry column chromatography on silicagel (MeOH in chloroform, 0 to 5%). The desired product was obtained ascolorless oil (169 mg, 0.18 mmol, 52%). 1HNMR (400 MHz, CDCl3) δ:4.03-3.95 (m, 6H), 2.55 (t-like, 7.1 Hz, 2H), 2.44 (quintet-like, 5.4Hz, 1H), 2.34-2.28 (m, 6H), 1.69-1.58 (m, 8H), 1.52-1.42 (m, 3H),1.42-1.17 (m, 70H), 0.92-0.87 (m, 18H), 0.82-0.70 (br., 1H).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, includingU.S. Provisional Patent Application No. 62/547,043, filed Aug. 17, 2017,are incorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description. In general, in the followingclaims, the terms used should not be construed to limit the claims tothe specific embodiments disclosed in the specification and the claims,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such claims are entitled.Accordingly, the claims are not limited by the disclosure.

The invention claimed is:
 1. A compound having the following structure(I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,wherein: X is N, and Y is absent; or X is CR, and Y is NR; L¹ is—O(C═O)R¹, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹, —C(═O)SR¹,—SC(═O)R¹, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c), —NR^(a)C(═O)NR^(b)R^(c),—OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹; L² is —O(C═O)R², —(C═O)OR²,—C(═O)R², —OR², —S(O)_(x)R², —S—SR², —C(═O)SR², —SC(═O)R²,—NR^(d)C(═O)R², —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f),—OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or a direct bond to R²; L³ is—O(C═O)R³ or —(C═O)OR³; G¹ and G² are each independently C₂-C₁₂ alkyleneor C₂-C₁₂ alkenylene; G³ is C₁-C₈ alkylene, C₂-C₈ alkenylene, C₁-C₈heteroalkylene or C₂-C₈ heteroalkenylene when X is CR, and Y is NR; andG³ is unsubstituted C₁-C₈ heteroalkylene or unsubstituted C₂-C₈heteroalkenylene when X is N, and Y is absent; R^(a), R^(b), R^(d) andR^(e) are each independently H or C₁-C₁₂ alkyl or C₁-C₁₂ alkenyl; R^(c)and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl; each Ris independently H or unsubstituted C₁-C₁₂ alkyl; R¹, R² and R³ are eachindependently C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl; and x is 0, 1 or
 2. 2. Thecompound of claim 1, wherein G³ is C₁-C₈ alkylene.
 3. The compound ofclaim 1, wherein G³ is unsubstituted C₁-C₈ heteroalkylene.
 4. Thecompound of claim 3, wherein G³ is C₁-C₈ aminylalkylene.
 5. The compoundof claim 1, wherein X is N and Y is absent.
 6. The compound of claim 1,wherein X is CR and Y is NR.
 7. The compound of claim 1, having one ofthe following structures (IA), (IB), (IC) or (ID):


8. The compound of claim 1, wherein L¹ is —O(C═O)R¹, —(C═O)OR¹ or—C(═O)NR^(b)R^(c), and L² is —O(C═O)R², —(C═O)OR² or —C(═O)NR^(e)R^(f).9. The compound of claim 8, wherein L¹ is —(C═O)OR¹ and L² is —(C═O)OR².10. The compound of claim 1, wherein L³ is —(C═O)OR³.
 11. The compoundof claim 1, wherein G¹ and G² are each independently C₄-C₁₀ alkylene.12. The compound of claim 1, wherein R¹, R² and R³ are each,independently, branched C₆-C₂₄ alkyl.
 13. The compound of claim 12,wherein R¹, R² and R³ each, independently have the following structure:

wherein: R^(7a) and R^(7b) are, at each occurrence, independently H orC₁-C₁₂ alkyl; and a is an integer from 2 to 12, wherein R^(7a), R^(7b)and a are each selected such that R¹ and R² are each independentlybranched and independently comprise from 6 to 20 carbon atoms.
 14. Thecompound of claim 13, wherein a is an integer from 8 to
 12. 15. Thecompound of claim 13, wherein at least one occurrence of R^(7b) is C₁-C₈alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,tert-butyl, n-hexyl or n-octyl.
 16. The compound of claim 1, wherein Xis CR, Y is NR and R³ is C₁-C₁₂ alkyl.
 17. The compound of claim 1,wherein R¹, R² and R³ independently have one of the followingstructures:


18. The compound of claim 1, having one of the following structures:


19. A composition comprising the compound of claim 1 and a therapeuticagent.
 20. The composition of claim 19, wherein the therapeutic agentcomprises a nucleic acid.
 21. The composition of claim 20, wherein thenucleic acid is selected from antisense and messenger RNA.
 22. A methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing the composition of claim 19,and administering the composition to the patient.
 23. A lipidnanoparticle comprising the compound of claim
 1. 24. A pharmaceuticalcomposition comprising the lipid nanoparticle of claim 23 and apharmaceutically acceptable diluent or excipient.